Adoptive T cell immunotherapy provides an attractive alternative to the current regime of antiretroviral medication for the treatment of HIV disease because it has the potential to be highly specific, long lasting, and free of significant side effects. Key to the success of adoptive T cell therapy is survival and retained functional capabilities of the T cells once they are re-infused into the host. The ex vivo expansion phase of adoptive T cell immunotherapy provides an opportunity to genetically engineer T cells to modify their resistance to HIV infection once re-infused into the host. The central hypothesis of this application is that T cells can be engineered so that they are immune to HIV entry and integration in vivo. We will undertake two approaches to engineer T cells to become resistant to HIV entry and integration. The first will make a multi-targeted lentiviral vector that will encode a humanized version of the rhesus TRIMSa, a cell based T-20-like fusion inhibitor, and/or a small hairpin (sh) RNA that targets CCR5 mRNA. We will employ both in vitro and in vivo HIV challenge assays as well as T cell functional assays to identify the best combination of antiviral agents. The second approach attempts to replicate the innate high resistance to HIV infection observed in individuals who are homozygous for a 32 base pair deletion in their CCR5 gene. By using zinc finger nucleases (ZFNs) specific to CCR5 we will induce double strand breaks in the CCR5 coding region. These double strand breaks will invoke the highly error prone non-homogologous end joining repair mechanism to make mutations that disrupt the production of functional CCR5. It is our hypothesis that T cells engineered in this manner we will have a competitive advantage in repopulating immunocomprised HIV infected individuals and serve to fight both HIV and opportunistic infections. This hypothesis will be tested in a Phase I clinical trial described in Project 1.